Design of Transmembrane Mimetic Structural Probes to Trap Different Stages of γ-Secretase-Substrate Interaction

Abstract

ABSTRACTγ-Secretase, a membrane-embedded aspartyl protease complex with presenilin as the catalytic component, cleaves within the transmembrane domain (TMD) of its many substrates, which include the amyloid precursor protein (APP) of Alzheimer’s disease (AD). APP TMD is processively cut by γ-secretase through endoproteolysis followed by tricarboxypeptidase “trimming”, the latter function being deficient with mutations causing hereditary AD. Toward better understanding of substrate recognition and hydrolytic reactions catalyzed by this enzyme within its hydrophobic milieu, we recently developed a prototype substrate TMD mimetic as a chemical tool for structural analysis (Bhattarai S et al. J. Am. Chem. Soc., 2020, 142(7): 3351-3355). This TMD mimetic—composed of a helical peptide inhibitor (HPI) connected through a linker to a transition-state analog inhibitor (TSA)— simultaneously engages the substrate docking exosite and the active site and is pre-organized to trap the protease transition state of carboxypeptidase trimming. In this study, we developed variants of this prototype designed to allow visualization of transition states for endoproteolysis, TMD helix unwinding, and lateral gating of substrate. New HPI-TSA conjugates were synthesized using an earlier established divergent strategy, which involved the construction of a tripeptidomimetic building block followed by solid-phase peptide synthesis (SPPS). For each class of structural probe, SAR analysis led to discovery of highly potent prototypes with stoichiometric or low-nanomolar inhibition. These TMD mimetics exhibited non-competitive inhibition of γ-secretase activity and occupy both docking and active site as demonstrated by enzyme cross competition experiments and photoaffinity probe binding assays. The new probes should be important structural tools for trapping different stages of substrate recognition and processing via ongoing cryo-electron microscopy with γ-secretase, ultimately aiding rational drug design.Abstract Graphic